The primary goal is to identify and analyze new genes in Drosophila that control biological rhythms or are regulated by the circadian clock. Both genetic and biochemical strategies will be employed. A large collection of chemically mutagenized strains will be screened; almost all of them harbor mutations (on the major autosomes) that have never been tested for effects on rhythms. In addition, these flies will be assayed in clock-mutant genetic backgrounds (an enhancer-suppressor approach), to increase sensitivity of the screen. A search will be initiated to induce novel mutations on the X and 4th chromosomes, which are not saturated or unexamined for genes influencing Drosophila rhythms. A mosaic strategy will be innovated to recover induced mutations that affect rhythmicity but are whole-organismal lethals. This strategy will take advantage of a set of transgenes to generate flies with mutant brain cells only in putative pacemaker neurons, which will deepen our understanding of the neural substrates of behavioral rhythmicity as well as generate novel mutants. In the molecular side of the project, the results of recent microarray and differential - display will be exploited to identify additional clock-controlled genes and to define the levels at which they are clock-regulated. This will entail the analysis of rhythm-mutant strains as well as specific cells and tissues. The latter will involve the development of new methods for assaying small amounts of RNA. A tissue culture approach will be exploited to identify the direct targets of several clock-relevant transcription factors. Polyribosomal mRNA will be isolated to investigate the possibility that translation is subject to circadian regulation. All cycling RNAs of interest and all novel rhythm mutants will be characterized with genetic, cellular, and biochemical tools, to define where and how they function in the fly's circadian system. Finally, microarrays will be applied to search for molecular circadian rhythms in C. elegans and in two species of yeast. Cycling gene expression is an ideal means to identify the elementary existence of circadian systems in these organisms, as it makes no assumptions about which physiological or biochemical systems are under circadian control. Moving beyond the currently appreciated evolutionary horizon of chronobiology is proposed in the spirit of many Drosophila clock genes that proved to have relatives in other species-including Homo sapiens, in which these factors connect to certain human maladies.